JP2004231902A - Antioxidant composition - Google Patents

Antioxidant composition Download PDF

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JP2004231902A
JP2004231902A JP2003025210A JP2003025210A JP2004231902A JP 2004231902 A JP2004231902 A JP 2004231902A JP 2003025210 A JP2003025210 A JP 2003025210A JP 2003025210 A JP2003025210 A JP 2003025210A JP 2004231902 A JP2004231902 A JP 2004231902A
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carnosine
anserine
antioxidant
mixture
membrane
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JP4833502B2 (en
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Nobuya Yanagiuchi
延也 柳内
Shigenobu Shiotani
茂信 塩谷
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Japan Research & Development Association For New Functional Foods
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Japan Research & Development Association For New Functional Foods
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Abstract

<P>PROBLEM TO BE SOLVED: To provide an antioxidant composition for an active oxygen species which has a strong antioxidative activity to a specific active oxygen species. <P>SOLUTION: The antioxidant composition comprises 100 mg of an anserin-carnosin mixture, 20-100 mg of vitamin C (L-ascorbic acid or L-sodium ascorbate), and 2.5-30 mg of vitamin E (α-tocopherol), wherein the anserin-carnosin mixture has an anserin-carnosin ratio of 3:1-1:3 adjusted by mixing various animal extracts extracted with hot water from one or more muscles of poultry, meat, and migratory fish, The composition has an antioxidative action specific for a hydroxy radical, a hypochlorite radical, and a peroxide nitrate radical. An antioxidant food composition comprises the antioxidant composition. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【0001】
【発明の属する技術分野】
本発明は、動物性天然エキスに由来する抗酸化能を有する特定の成分を特定の量比で配合してなる抗酸化用組成物に関するものであり、更に詳しくは、特定の活性酸素種に対して有効に抗酸化能を発揮できる当該活性酸素種抗酸化用組成物に関するものである。本発明は、人体内で発生する活性酸素による酸化ストレスを解消して広義の生活習慣病の予防、具体的には体内活性酸素が顕著に高い糖尿病患者の糖尿病関連疾患の予防に供せる食品及び食品素材の創製を可能とするものとして有用である。
【0002】
【従来の技術】
近年、わが国では、高齢者人口の増加に伴う各種成人病の増加とその医療に関わる人的、経済的負担の増大が極めて深刻な問題として取り上げられている。生物の老化と病気の発生は言わば不可分の関係にあるので、高齢者が増加すれば病人の数も増えることは避けられないことである。そこで、人間の老化する速度を可能な限り遅くし、病気の発生もなるべく抑制しようということが重要になる。人間が老化する第一の原因は、体を構成する細胞には分裂増殖する回数に限りがあることであり、細胞が傷害を受けることによって細胞の老化が促進されることにある。細胞を傷害する要因として、紫外線や放射線の照射、薬物による有害作用などがあるが、最も大きい要因は、生体を構成する成分が酸化されることであると言われている。
【0003】
この酸化反応を起こす物質は、活性酸素と呼ばれるものであるが、活性酸素は、蛋白質、糖、脂肪などの生体構成成分を酸化する以外に、細胞の遺伝子DNAを直接傷害する作用を持っている。しかも、活性酸素は、我々人間が食物として体内に持ち込むものは少なく、酸素を利用してエネルギーを生産している我々の体内で毎日生産されているものであり、紫外線や放射線を浴びても活性酸素が体内に生産されるし、タバコや排気ガスとともに活性酸素も吸い込む。また、体内に侵入した細菌を殺菌するために、白血球は活性酸素を作り出す酵素を分泌して活性酸素を放出し、精神的なストレスや病気などでも体内の活性酸素は増加する。これらの活性酸素は、必ず細胞のDNAを傷害して細胞の老化を促進し、時には細胞を突然変異させて異常細胞やガン細胞の発生を促進することにもなるのである。特に注目されることは、糖尿病患者の生体内では正常人に比較して活性酸素が顕著に高まることであり、糖尿病に関連する疾患として、糖尿病性動脈硬化、高血圧、末梢循環障害、腎症などが知られているが、このことは、これらの典型的な生活習慣病の発生に、この活性酸素が密接に関与していることを示していることである。
【0004】
このように、あらゆる細胞の老化と病気の発生に活性酸素が関与していることから、人間の老化を抑え、可能な限り健康を維持させるために、活性酸素を消去する抗酸化剤が重要視されて来た。従来、抗酸化性ビタミンと呼ばれるビタミンC(V.Cと略記)やビタミンE(V.Eと略記)が典型的な活性酸素消去剤として広く使用されているが、これに加えて、最近では、緑黄色野菜のβ―カロチン、お茶や大豆に含まれるカテキン類、フラボノイド化合物、ブドウの種子に含まれるイソチオシァネートなどが注目されて来た。また、本発明で利用する動物組織に存在するヒスチジン含有ジペプチドのアンセリンやカルノシンも有力な抗酸化性物質として関心が持たれて来た。
【0005】
ところで、ここに挙げた抗酸化性物質のうち、アンセリンやカルノシンを除いて、V.C、V.E、ポリフェノール類などは、全て植物由来のものである。抗酸化性物質の素材として植物原料が多い理由は、植物は太陽光線を浴びて生育し、かつ酸素も利用しているから、植物の生体内で発生する活性酸素の量は極めて多く、また、酸化されやすい脂質の合成も活発に行うからである。一方、動物の場合にも植物と同様に生体内で抗酸化作用を持つ物質を生産する能力があり、例えば、貝類が作るアミノ酸の一種のタウリン、脊椎動物の筋肉組織で作られる上記のアンセリン−カルノシンがそれである。我々人間にとって、これらの動物由来の抗酸化性物質が有益なのか、植物由来のものが有益なのかは確たる知見はない。
【0006】
植物性抗酸化剤であるV.CとV.Eは、人間が利用でき、かつ必須なものである。しかし、これらを含めて植物性抗酸化剤が動物生体内で発生するどのような活性酸素に有効で、どのようなものには無効か、更にはこれらの植物由来抗酸化性物質がどのように人間の腸から吸収され、人間の体内で有効に抗酸化作用を発揮しているのか否かは、完全に解明されているわけではない。また、動物由来のタウリンやアンセリン−カルノシンは、生体内で生成され、腸からの吸収も植物性抗酸化剤とは比較にならないほど高いが、しかし、植物性抗酸化剤ほどには抗酸化性食品素材としての利用は進んでいなかった。
【0007】
これまで、天然の抗酸化性物質の抗酸化活性評価には、活性酸素と同様に分子内にラジカルを持つDPPH色素(1,1’−Diphenyl−2−picrylhydrazyl,非特許文献1参照)を捕捉する作用で評価する方法や、同じくラジカル発生物質と反応して蛍光を発するルミノールの蛍光発光消去作用で評価する方法(2,2’−azobis−2−amidinopropane,非特許文献2参照)が用いられて来た。しかし、これらの試験で用いられるDPPHラジカル色素やルミノールを酸化する反応は我々の生体内で起こる酸化反応ではない。従って、これらの方法で強い抗酸化活性が認められても、それがそのまま生体内で有効であるとは限らないのである。また、活性酸素には、様々な物質が存在しているので、DPPH法や蛍光発光消去法で強い活性を有する抗酸化性物質が生体内の全ての活性酸素に対して有効に働いているという証拠もない。更に、植物由来の抗酸化性物質の多くは、V.Eを含めて多環芳香族化合物からなり水に溶解しないものがほとんどであり、人間がこれらの物質を吸収し、利用するためにはそれらを運ぶ脂質蛋白質等の共存が必須である。
【0008】
脊椎動物原料の熱水抽出エキス中には、ヒスチジンを含有する抗酸化性ジペプチド(Histidine−containg dipeptide, HCDPと略記)であるアンセリン(β−alanyl−1−methyl−L −histidine)とカルノシン(β−alanyl− L−histidine) が含まれているが、V.CやV.Eが植物由来の抗酸化性物質であるのに対して、これらは動物に由来する抗酸化性物質である。この動物由来アンセリン−カルノシンは、抗酸化剤として様々な生理機能が知られており、老化防止作用や糖尿病関連疾病などの予防効果も期待されている。しかし、抗酸化剤としてのこれらの実用的な利用は植物性抗酸化剤ほどには普及していなかった。この理由の一つとして、アンセリンやカルノシンをDPPH法や蛍光発光法で抗酸化活性を測定すると、後記する表1に示されるように、植物由来のV.CやV.Eに比べていずれも1万分の1以下に測定されるので、抗酸化性物質としての意義を見出すことが困難であったことが挙げられる。
【0009】
【非特許文献1】
T. Yamaguchi, et al. Biosci. Biothech. Biochem. 62巻、1201−1204頁、1998)
【非特許文献2】
O. Hirayama, et al. Analytical Biochemistry 、247巻、237−241頁、1997年
【非特許文献3】
A.R.Hipkiss ら、Cellular and Molecular Life Sciences,57巻、747−753頁、2000年
【0010】
これまでに、抗酸化性食品と呼ばれるものは、V.CやV.Eをはじめ植物由来の抗酸化剤を含有する極めて多種・多様なものが生産、販売されているが、それらの抗酸化活性が主として上記のDPPH法や蛍光発光法などによる評価であったために、生体内で有効に抗酸化活性を示す証拠に乏しいものであった。しかも、ポリフェノール類と総称される植物由来の多環芳香族化合物は吸収性の問題やこれらを運ぶ脂質や脂質蛋白質の共存が不可欠であり、動物の生体内で有効に抗酸化活性を発揮するか否かは依然不明なことが多かった。これらのことから、幅広い活性酸素種に対して有効に作用することを明示した抗酸化性食品や抗酸化性成分を組み合わせた食品組成はこれまでのところ皆無である。
【0011】
【発明が解決しようとする課題】
このような状況の中で、本発明者らは、幅広い活性酸素種に対して有効に抗酸化作用を発揮する当該活性酸素種抗酸化用組成物を開発することを目標として鋭意研究を進める過程で、チキンエキスやその他の動物エキスに含まれる抗酸化性HCDPであるアンセリンとカルノシンに着目し、エキス自身の風味や旨みを保持させたままそのHCDP含有量に見合った量の植物由来抗酸化性ビタミンであるV.CとV.Eを補強することによって、幅広い活性酸素に対して作用し、かつ生体蛋白質がこれらの活性酸素により酸化分解されることを防止できることを見出し、本発明を完成するに至った。
本発明は、水酸化ラジカル、ジ亜塩素酸ラジカル及び過酸化硝酸ラジカル等の活性酸素種に対して特異的な抗酸化作用を有する当該活性酸素種抗酸化作用組成物、及び当該抗酸化用組成物を配合した抗酸化性食品組成物を提供することを目的とするものである。
【0012】
【課題を解決するための手段】
上記課題を解決するための本発明は、以下の技術的手段より構成される。
(1)食用家禽類、畜肉類、及び回遊性魚類の1種以上の筋肉より熱水抽出して得られる動物性エキス中のアンセリン−カルノシン混合体の、アンセリンとカルノシンの含量比率を任意に各種エキスを混合することによって、3:1〜1:3の範囲になるように調整し、該アンセリン−カルノシン混合体100mgに対してビタミンC(L−アスコルビン酸又はL−アスコルビン酸ナトリウム)を20〜100mg、ビタミンE(α−トコフェロール)を2.5〜30mgの割合で配合したことを特徴とする水酸化ラジカル、ジ亜塩素酸ラジカル及び過酸化硝酸ラジカルに対して特異的な抗酸化作用を有する当該活性酸素種抗酸化用組成物。
(2)上記アンセリン−カルノシン混合体が、上記熱水抽出して得られる動物エキスを分画分子量3,000〜10,000の限外ろ過膜処理して、ろ過膜透過画分として得られるアンセリン−カルノシン混合体であることを特徴とする前記(1)記載の抗酸化用組成物。
(3)上記アンセリン−カルノシン混合体が、上記熱水抽出して得られる動物エキスを分画分子量3,000〜10,000の限外ろ過膜処理して、該ろ過膜透過液を更に分画分子量200〜500のナノろ過膜もしくは逆浸透膜で処理してろ過膜上に透過阻止されて濃縮されたアンセリン−カルノシン混合体であることを特徴とする前記(1)記載の抗酸化用組成物。
(4)上記アンセリン−カルノシン混合体が、上記熱水抽出して得られる動物エキスを分画分子量3,000〜10,000の限外ろ過膜処理して、該ろ過膜透過液を更に分画分子量200〜500のナノろ過膜もしくは逆浸透膜で処理してろ過膜上に濃縮されたものを陽イオン交換体クロマトグラフによって純度75%以上に精製されたアンセリン−カルノシン混合体であることを特徴とする前記(1)記載の抗酸化用組成物。
(5)前記(1)から(4)のいずれかに記載の抗酸化用組成物を配合したことを特徴とする抗酸化性食品組成物。
【0013】
【発明の実施の形態】
次に、本発明について更に詳細に説明する。
本発明者らは、従来の食品素材として得られる抗酸化性物質が生体内で有効に抗酸化活性を示すか否かを評価するために、実際に生体内で発生する活性酸素を用いて、それらの動物蛋白質を酸化分解する反応に対して抗酸化性物質がどのように作用するか、そして、その抗酸化作用の強さはどのようなものであるかを試験してみた。
生体内で発生する代表的な活性酸素としては、過酸化水素(H )、水酸化ラジカル(OH・)、ジ亜塩素酸ラジカル(ClO・)、過酸化硝酸ラジカル(ONOO・)などがある。そこで、試験管内で蛋白質を酸化分解する能力の高いOH・、ClO・、及びONOO・の3種類を作製し、試験に供した。また、各抗酸化剤の抗酸化活性の比較では、ヘモグロビン、卵白アルブミン、血清アルブミンを標的蛋白質として用いた。その結果、V.C、V.E及びアンセリン−カルノシンが示す抗酸化活性は、活性酸素の種類によって大きく異なり、V.CはONOO・に対して、V.EはOH・に対して、そして、アンセリン−カルノシンはClO・に対してそれぞれ最も強い抗酸化活性を示すことが分かった。アンセリンとカルノシンの特徴を更に挙げると、アンセリンはOH・に対してカルノシンより強く、逆にカルノシンはClO・に対してアンセリンよりも強い抗酸化性を示すことが分かった。これらの結果を総合して抗酸化活性を評価すると、後記する表2に示されるように、DPPH法や蛍光発光法による評価とは全く異なり、動物由来の抗酸化性物質であるアンセリン−カルノシンは、動物蛋白質であるヘモグロビンや卵白アルブミン、牛血清アルブミンの酸化分解に対してV.CやV.Eとほぼ同等の抗酸化活性を持っていることが判明した。
【0014】
本発明者らが調べたところ、チキンエキス中にはアンセリン−カルノシンが2:1〜4:1の比率で含有しており、牛肉エキスでは1:3〜1:5、豚肉エキスでは1:4〜1:10、マグロエキスでは10:0〜20:1の比率で存在していること、これらのアンセリンとカルノシンはClO・とONOO・ラジカルに対して最も強い抗酸化活性を示すが、OH・ラジカルではV.Eより弱く、V.Cと類似の抗酸化作用を有していること、また、アンセリンはOH・に対してカルノシンより強い抗酸化作用を持つが、カルノシンはClO・とONOO・に対してアンセリンよりも強い抗酸化作用を持っていること、そして、これらのことから、HCDP中に占めるアンセリン−カルノシン混合体のアンセリンとカルノシンの含量比率を任意に各種エキスを混合することによって、3:1〜1:3の範囲になるように調整し、更に総量として測定されるアンセリン−カルノシン100mgに対してV.Cを20〜100mg、V.Eを2.5〜30mgの割合で配合することにより、これらの3種の系統の異なる活性酸素による蛋白質酸化をほぼ完全に阻止することが可能であることが分かった。
【0015】
また、天然エキスの味や旨みを生かした各精製段階のアンセリン−カルノシン混合体でも、HCDP含量として調整すれば高純度アンセリン−カルノシンと同等の抗酸化活性が得られるので、各種原料から抽出された天然エキスの状態のままでも、このV.CとV.Eの添加量で高い抗酸化活性を持ったエキスを作製することが可能である。なお、本発明に係る天然エキス中のアンセリン−カルノシン含有比率が適切な範囲から逸脱するものは、例えば、豚肉エキスとマグロ、カツオなどの魚肉エキスであり、豚肉の場合はカルノシン含量が高く、魚肉エキスではアンセリン含量が高い。これらのエキスは、豚肉エキスと魚肉エキスから得られるHCDPをほぼ1:1に混合することによって抗酸化活性を発現させる上で適切なアンセリンとカルノシンの含有比率3:1〜1:3の範囲にすることが可能である。
【0016】
動物性エキス中に存在するアンセリン−カルノシンの抗酸化剤としての有用性や生活習慣病予防効果の可能性についてこれまで多くのことが報告されて来た。しかし、植物性抗酸化剤ほどには一般の実用が進んでいなかったが、その原因として、一般的に使用される抗酸化活性測定法において、V.CやV.Eなどの抗酸化性ビタミン類より極めて低い活性しか示さなかったことが挙げられる。しかし、生体内には数多くの活性酸素が生成され、これらが直接、老化や疾病の原因になっているのに対して、従来の方法では抗酸化剤のこれらに対する作用様式は不明であった。本発明によれば、生体内で発生する代表的な活性酸素3種類のClO・、ONOO・及びOH・に強く作用する抗酸化性組成物が得られるが、従来の抗酸化剤を組み合わせてもこのような効果は得られない。これらは、従来の評価法による結果と異なるものであり、従来の評価法では知り得なかったことである。本発明の組成物は、上記成分を特定の量比で組み合わせた場合に、上記活性酸素種に対して特異的、かつ相乗的な抗酸化作用を示すものであり、これらの構成及びその有効性は、前記試験例及び実施例に示した格別の実験を実施することではじめて実証されたものであり、その作用効果は従来の知見からは予期し得ないものである。従来の生活習慣病や老化、ガンの発生を予防しようという目的に、V.CやV.E、或はアンセリンとカルノシンを単独で使用した場合には、いずれも生体内で発生する活性酸素に対して有効ではなく、活性酸素の種類によっては全く無効の場合があった。しかし、本発明の組成物によれば広い種類の活性酸素に対して有効に抗酸化活性を発揮でき、従って、従来には無い新規な生活習慣病や疾病予防に有効な抗酸化性組成物を調製し、提供することが可能である。
【0017】
次に、試験例を示して本発明を詳細に説明する。
試験例1
(1)従来法によるアンセリン−カルノシンの抗酸化活性
動物性エキス中の抗酸化性ペプチドであるアンセリンとカルノシンを植物性抗酸化ビタミンであるV.CとV.Eの抗酸化活性を比較するために、上記のDPPH法とケミルミネッセンス法(蛍光発光法)で試験した。アンセリン・硝酸塩とカルノシンは試薬(シグマ社製)を用い、動物性エキス中のアンセリン−カルノシン混合体は各精製段階のチキンエキスを用いた。V.CとしてはL−アスコルビン酸及びL−アスコルビン酸ナトリウム試薬(和光純薬製)を使用し、また、V.Eとしてはα−トコフェロールをショ糖エステルに乳化させて親水性を付与した超エマルジョン(商品名:サンアクティブVE−202、VE−702、太陽化学製)を用いた。その結果は表1に示す通りであった。DPPH法と蛍光発光法ではいずれもV.Cが最も強い抗酸化活性を示し、次いで、V.Eであり、試薬のアンセリンとカルノシン、精製したアンセリン−カルノシン混合体はいずれも弱い抗酸化活性として測定された。
【0018】
【表1】

Figure 2004231902
【0019】
(2)蛋白質酸化分解系でのアンセリン−カルノシンの抗酸化活性
人間の生体内で発生する代表的な活性酸素4種のうち、H は蛋白質を酸化分解する作用が低く判定が困難であることから、その他のOH・、ClO・及びONOO・の3種を用いて試験した。OH・はFenton法(B.Halliwellら、Analytical Biochemistry 、165巻、215〜219頁、1987年の改変)、ClO・はジ亜塩素酸ナトリウム、ONOO・はquenching flow reactor法(R.Radi ら、Journal of Biological Chemistry、266巻、4244〜4250頁、1991年)で調製した。蛋白質を2.5mg/mLになるように緩衝化生理食塩水、pH7.2に溶解し、蛋白溶液に対して10:1の比率で各濃度の活性酸素(ClO・:400ppm、OH・:800ppm、ONOO・:5mM)を添加して、37℃、30分間反応させた。また、抗酸化性物質は活性酸素添加の30分前に蛋白溶液に対して同じく10:1の比率で添加した。各反応液を、SDS−ポリアクリルアミドゲル電気泳動法(SDS−PAGE)で泳動した後、染色したゲルをデンシトメーターで、或は蛋白質反応液を直接TSKG−3,000SWカラムのGPC−HPLCにかけて、蛋白分解阻止活性を次の式で算出した。
【0020】
蛋白質分解阻止活性(%)=抗酸化剤5mM添加時の活性酸素分解蛋白質のピーク面積 ×100(%)/未分解蛋白質のピーク面積
【0021】
その結果を表2に示す。表2の結果から、ClO・とONOO・に対しては各種エキス中に含まれるアンセリンとカルノシンが最も抗酸化活性が強く、V.Cはアンセリンとカルノシンに類似の抗酸化活性を示し、V.EはOH・に対して最も強い抗酸化活性を示すことが分かった。
【0022】
【表2】
Figure 2004231902
【0023】
(3)抗酸化剤の配合による活性酸素の蛋白質分解阻止作用
チキンエキスなどの動物エキス中に含まれるアンセリンとカルノシンは、生体内で発生する活性酸素に対してV.Cに類似した抗酸化作用の傾向を持っているが、V.Eとは対照的である。従って、V.Eが最も強く、動物由来のアンセリン−カルノシン混合体やV.Cが最も弱いOH・に対して、そして、動物由来アンセリン−カルノシン混合体が最も強く、V.Eが最も弱いClO・に対してこれらは相互補完的な効果が期待でき、更に、ONOO・ではアンセリン−カルノシン混合体にV.CとV.Eを加えることによって更に完全な抗酸化活性が期待できる。各種エキス由来のアンセリン−カルノシン混合体の濃度を1.2mg/mL(約5mM)とし、V.Cナトリウムを0.6mg/mL(約3.0mM)、V.Eを0.5mg/mL(約1mM)添加配合した場合の蛋白分解阻止作用を表3に示す。
【0024】
【表3】
Figure 2004231902
【0025】
表3に示した結果の通り、ポークエキスとマグロエキスではV.CとV.Eを添加してもClO・による酸化分解を完全に阻止することができなかったが、これは、表4に示すように、アンセリンとカルノシンの含有に偏りがあったためと考えられた。表3と表4の結果から、HCDPが適切な抗酸化活性を発揮するためのアンセリンとカルノシンの含量比率は、チキンエキスの比率とビーフエキスの比率の範囲である。すなわち、アンセリン−カルノシンが3:1〜1:3の範囲であった。実施例3で調製したUF処理ビーフエキス、同ポークエキス、同マグロエキスを等量混合した場合、アンセリンとカルノシンの含有比率は1.8:1.7とほぼ等量となったが、この混合エキスにV.CとV.Eを添加したもののClO・に対する酸化分解阻止率は100%であった。
【0026】
【表4】
Figure 2004231902
【0027】
なお、アンセリン−カルノシン混合体の濃度を2.4mg/mL(約10mM)にした場合には、V.CとV.Eの添加量をそれぞれ0.5mg/mL(約2.5mM)、0.06mg/mL(約0.15mM)でOH・酸化分解に対して95%以上の分解阻止活性を示す結果であった。すなわち、動物エキス由来のアンセリン−カルノシン混合体の量を増加させることによって抗酸化活性は上昇するが、アンセリン−カルノシンが完全に抑制できないOH・ラジカルに対しては、アンセリン−カルノシンとの重量比でV.Cを5:1、V.Eを40:1の割合で添加すると各種活性酸素による蛋白質分解は十分に抑制される結果が得られた。これは、アンセリン−カルノシン混合体100mg当たりV.Cは20mg、V.Eは2.5mgとなる。また、上限値は各抗酸化剤の推奨される一日最大摂取量となるので、アンセリン−カルノシンとV.Cは3,000mg、V.Eは300mgであることから、アンセリン−カルノシン100mg当たりV.Cは100mg、V.Eは30mgの割合となる。
【0028】
(4)糖尿病ラット(GKラット)における糖化蛋白質生成抑制作用
糖尿病の病態では血中の活性酸素量が増加し、酸化された蛋白質と血中の高ブドウ糖値により糖化蛋白質、具体的には糖化モグロビンA1c量が増加することが知られている。この活性酸素の増加に伴う糖化蛋白質の生成は最終糖化産物(Advanced glycation endoproducts、AGEsと略記)の生体内蓄積を意味し、糖尿病に併発する各種の疾患、すなわち、糖尿病性高血圧、同動脈硬化、同腎症、同痴呆症、同末梢循環障害などがこのAGEsの蓄積に起因していると考えられている。過剰栄養摂取により発症する2型糖尿病のモデル動物であるGKラット(Y.Iharaら、FEBS Letters、473巻、24−26頁、2000年)に対して、本発明に関わるV.C及びV.Eを添加したアンセリン−カルノシン混合体が実際に生体内の酸化反応により促進される糖化ヘモグロビンA1c生成の抑制作用を持つか否かを試験した。図3に示すとおり、GKラットの体重1kg当たりUF処理チキンエキスのアンセリン−カルノシン200mg、V.C100mg、V.E20mgを経口投与した場合、上記Y.Iharaらにより報告されたV.Eの20mg単独投与より、アンセリン−カルノシン200mgを含む飼料を投与したほうが明らかに糖化ヘモグロビンA1c値が低下する結果が得られた。
【0029】
【実施例】
次に、実施例に基づいて本発明を具体的に説明するが、本発明は、以下の実施例によって何ら限定されるものではない。
実施例1
丸鶏から熱水抽出して得られたチキンエキス40Lを分画分子量3,000
の限外ろ過膜(旭化成製SEP−1013)にかけ、ろ過膜透過液32Lを回収した。ろ過液を減圧濃縮してBrix濃度20%にした。この濃縮チキンエキスのアンセリン−カルノシン含量は固形物当たり180mg/gであったので、V.C(食品添加物)を90mg/g、V.E(太陽化学製サンアクティブVE−202)を18mg/gになるように添加し、更にデキストリン(食品添加物)を濃縮チキンエキスの固形物当たり33%(1:2)になるように加えて噴霧乾燥し、抗酸化性チキンエキス粉末約30gを調製した。この粉末は1g当たりアンセリン−カルノシンを120mg、V.Cを60mg、V.Eを12mg含有しており、上記のGKラットに2週間経口投与すると図3に示したとおり、血中糖化ヘモグロビン含量を低下させる活性を有していた。
【0030】
実施例2
丸鶏から熱水抽出したチキンエキス1,200Lを分画分子量3,000の限外ろ過膜(旭化成製SEP−3013)にかけ、ろ過膜透過液200Lを分画分子量500(Danish Separation Systems社製NFT−50)のナノろ過膜で濃縮して、Brix濃度10%のチキンエキス20Lを調製した。このナノろ過処理チキンエキスには固形物1g当たりアンセリン−カルノシンが200mg含有されているので、実施例1と同様に、V.CとV.Eを添加して凍結乾燥法により粉末化した。また、ナノろ過濃縮チキンエキス20Lを一度製造用水を加えて脱塩し、0.5Mリン酸ナトリウム緩衝液を4L加えて、0.1Mリン酸緩衝液、pH5.0で平衡化したDowex50WX−8陽イオン交換樹脂カラムに通液し、アンセリン−カルノシンをカラムに吸着させ、希薄な緩衝液でカラムを洗浄した後、アルカリ溶液でアンセリン−カルノシンを溶出させて回収した。この液を減圧濃縮してから活性炭で脱色し、エタノール沈澱法でアンセリン−カルノシンを沈澱させ、アルコールを減圧下に除去して精製されたアンセリン−カルノシン混合体粉末約10gを調製した。この精製粉末のアンセリン−カルノシン含量比率は1.2:1.0であり、2%レモン果汁100mL当たりアンセリン−カルノシンの総量が500mg、V.C250mg、V.E30mgになるように添加して抗酸化性清涼飲料を調製した。
【0031】
実施例3
骨を除去した豚肉、牛肉、マグロ肉1kg当たり3Lの水を加えて98℃、1時間熱水抽出し、分液ロートで油層を分離した後、珪藻土をろ過補助剤として吸引ろ過した。これらのエキス中にはアンセリンとカルノシンが混合体として含有され、固形分1g当たりのアンセリン−カルノシン含量は豚で96mg、牛で67mg、マグロで120mgであった。これらのエキスを減圧濃縮してBrix5.0%とし、3種を等量混合した。このときのアンセリン−カルノシン含量比率は1.8:1.7であったので、アンセリン−カルノシン混合体100mg当たりV.Cナトリウム(食品添加物)を50mg、V.E(太陽化学製、サンアクティブVE720)を20mg添加して無菌ろ過し、液状の動物性エキスを調製した。また、これらの珪藻土ろ過したエキスを限外ろ過膜(旭化成製SEP−1013)にかけて蛋白質を除去し、減圧濃縮して前記と同様に混合し、アンセリン−カルノシン混合体100mg当たりV.Cナトリウム50mg及びV.Eを30mg添加し、同様に液状の抗酸化性動物エキスを調製した。これらのエキスは表3に示した3種の活性酸素による蛋白質の分解をほぼ完全に抑制する活性を示すものであった。
【0032】
【発明の効果】
以上詳述したとおり、本発明は、動物性天然エキスに由来する抗酸化能を有する特定の成分を特定の量比で配合してなる抗酸化用組成物に係るものであり、本発明により、次のような効果が奏される。
(1)多様な活性酸素種に対して特異的な抗酸化作用を有する当該活性酸素種抗酸化用組成物を提供することができる。
(2)生体内で発生する代表的な活性酸素3種類の全てに抗酸化性能を有する新規抗酸化性組成物を提供することができる。
(3)従来、V.CやV.Eなどの抗酸化性ビタミン類より極めて低い活性しか示さないと考えられていたアンセリンとカルノシンを有効成分とする広い活性酸素種に対する抗酸化用組成物を提供することができる。
(4)アンセリン−カルノシン混合体、V.C、及びV.Eを組み合わせることで、それらの各成分単独では期待できない相乗的な抗酸化作用が得られる。
(5)これらのことより、活性酸素が関わる生活習慣病やその他の疾病の発症を効果的に予防できる抗酸化性組成物又は食品の調製が可能である。
【図面の簡単な説明】
【図1】図1は、生体内で発生する代表的な活性酸素による卵白アルブミンの酸化分解に対する各種抗酸化剤の防止作用を示す(A:ClO・、B:OH・、C:ONOO・、1:未分解卵白アルブミン、2:分解卵白アルブミン、3:V.C添加、4:V.E添加、5:アンセリン添加、6:カルノシン添加、7:チキンエキス由来精製アンセリン−カルノシン添加)。
【図2】図2は、糖尿病病態モデルのGKラットにおける本発明物質の血中糖化ヘモグロビンA1c生成抑制作用を示す(CE+VC+VE:UF処理チキンエキス+ビタミンC+ビタミンE;CE+VC:UF処理チキンエキス+ビタミンC;VE:ビタミンE単独)。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an antioxidant composition obtained by blending a specific component having an antioxidant ability derived from an animal natural extract in a specific quantitative ratio, and more specifically, to a specific active oxygen species. The present invention relates to the active oxygen species antioxidant composition which can effectively exhibit antioxidant ability. The present invention is intended to prevent oxidative stress caused by active oxygen generated in the human body to prevent lifestyle-related diseases in a broad sense, specifically, a food that can be used for prevention of diabetes-related diseases in diabetic patients whose active oxygen in the body is remarkably high, and It is useful as a material that enables creation of food materials.
[0002]
[Prior art]
In recent years, in Japan, the increase in various types of adult diseases associated with the increase in the elderly population and the increase in human and financial burden related to medical care have been taken up as extremely serious problems. Since the aging of living things and the occurrence of diseases are inseparable, it is inevitable that the number of sick people will increase as the number of elderly people increases. Therefore, it is important to reduce the rate of aging of humans as much as possible and to minimize the occurrence of diseases. The primary cause of aging of humans is that the cells that make up the body have a limited number of divisions and multiplications, and that aging of cells is promoted by damage to the cells. Factors that damage cells include irradiation with ultraviolet rays and radiation, adverse effects of drugs, and the like, but the biggest factor is said to be oxidation of components constituting the living body.
[0003]
The substance that causes this oxidation reaction is called active oxygen. Active oxygen has the effect of directly damaging cellular gene DNA in addition to oxidizing biological components such as proteins, sugars, and fats. . Moreover, active oxygen is rarely brought into the body as food by humans, and is produced daily in our body, which uses oxygen to produce energy, and is active even when exposed to ultraviolet rays or radiation. Oxygen is produced in the body, and it also inhales active oxygen along with tobacco and exhaust gases. In addition, in order to kill bacteria that have entered the body, leukocytes secrete enzymes that produce active oxygen and release active oxygen. Active oxygen in the body increases even when mental stress or illness occurs. These active oxygens inevitably damage the DNA of the cells and promote senescence of the cells, and sometimes mutate the cells to promote the development of abnormal cells and cancer cells. Of particular note is the fact that active oxygen is significantly increased in the body of diabetic patients compared to normal individuals, and diabetes-related diseases such as diabetic arteriosclerosis, hypertension, peripheral circulatory disorders, and nephropathy Is known, indicating that the active oxygen is closely involved in the development of these typical lifestyle-related diseases.
[0004]
Since active oxygen is involved in the aging of all cells and the development of diseases, antioxidants that eliminate active oxygen are important for suppressing human aging and maintaining health as much as possible. I have been. Heretofore, vitamin C (abbreviated as VC) and vitamin E (abbreviated as VE), which are called antioxidant vitamins, have been widely used as typical active oxygen scavengers. Attention has been focused on β-carotene, a green-yellow vegetable, catechins and flavonoid compounds contained in tea and soybeans, and isothiosinate contained in grape seeds. In addition, histidine-containing dipeptides such as anserine and carnosine which are present in animal tissues used in the present invention have been attracting interest as potential antioxidants.
[0005]
By the way, among the antioxidants listed here, except for anserine and carnosine, V.I. C, V. E, polyphenols, etc. are all derived from plants. The reason that many plant materials are used as a material for antioxidants is that plants grow in the sunlight and also utilize oxygen, so the amount of active oxygen generated in plants is extremely large, This is because the synthesis of lipids that are easily oxidized is also actively performed. On the other hand, animals have the ability to produce a substance having an antioxidant effect in vivo as well as plants. For example, taurine, an amino acid produced by shellfish, and the above-mentioned anserine produced by vertebrate muscle tissue. Carnosine is that. For us humans, there is no definitive knowledge whether these animal-derived antioxidants or plant-derived ones are beneficial.
[0006]
V. is a plant antioxidant. C and V. E is available and essential to humans. However, plant antioxidants, including these, are effective for any active oxygen generated in animal organisms, are not effective on what, and how these plant-derived antioxidants are effective. Whether it is absorbed from the human intestine and exerts an effective antioxidant effect in the human body has not been completely elucidated. In addition, animal-derived taurine and anserine-carnosine are produced in vivo, and their absorption from the intestine is incomparably higher than that of botanical antioxidants, but is not as antioxidant as botanical antioxidants. The use as food material did not progress.
[0007]
Until now, in the evaluation of the antioxidant activity of a natural antioxidant, a DPPH dye (1,1′-Diphenyl-2-picrylhydrazyl, see Non-Patent Document 1) having a radical in the molecule as well as active oxygen has been captured. And a method of evaluating the luminescence of luminol which emits fluorescence by reacting with a radical-generating substance (2,2'-azobis-2-amidinopropane, see Non-Patent Document 2). I came. However, the reaction that oxidizes the DPPH radical dye and luminol used in these tests is not an oxidation reaction that occurs in our living body. Therefore, even if a strong antioxidant activity is observed by these methods, it is not always effective in vivo as it is. Also, since various substances are present in active oxygen, it is said that antioxidant substances having strong activity in the DPPH method and the fluorescence emission elimination method are effectively acting on all active oxygen in the living body. There is no evidence. In addition, many plant-derived antioxidants are V. Most of the compounds, including E, are made of polycyclic aromatic compounds and do not dissolve in water. In order for humans to absorb and use these substances, the coexistence of lipid proteins and the like carrying them is essential.
[0008]
In the extract of hot water extracted from vertebrate, anserine (β-alanyl-1-methyl-L-histidine) and carnosine (β), which are histidine-containing antioxidant dipeptides (abbreviated as HCDP), are contained in hot water extract. -Alanyl-L-histidine). C and V. While E is a plant-derived antioxidant, they are animal-derived antioxidants. This animal-derived anserine-carnosine is known for various physiological functions as an antioxidant, and is also expected to have an antiaging effect and a preventive effect such as diabetes-related diseases. However, their practical use as antioxidants has not been as widespread as plant antioxidants. One of the reasons for this is that when the antioxidant activity of anserine or carnosine is measured by the DPPH method or the fluorescence emission method, as shown in Table 1 described below, plant-derived V. aureus and carnosine are determined. C and V. Since each of them is less than 1 / 10,000 as compared with E, it is difficult to find significance as an antioxidant.
[0009]
[Non-patent document 1]
T. Yamaguchi, et al. Biosci. Biotech. Biochem. 62, 1201-1204, 1998)
[Non-patent document 2]
O. Hirayama, et al. Analytical Biochemistry, 247, 237-241, 1997.
[Non-Patent Document 3]
A. R. Hipkiss et al., Cellular and Molecular Life Sciences, 57, 747-753, 2000.
[0010]
Until now, what is called antioxidant foods has been C and V. E and a very wide variety of plants containing plant-derived antioxidants are produced and sold, but their antioxidant activity was mainly evaluated by the above-described DPPH method and fluorescence emission method, etc. The evidence for effective antioxidant activity in vivo was poor. In addition, plant-derived polycyclic aromatic compounds collectively referred to as polyphenols are indispensable for the problem of absorptivity and the coexistence of lipids and lipid proteins carrying them, and do they exhibit effective antioxidant activity in the living body of animals? Whether or not was often unknown. From these facts, there has been no antioxidant food or food composition combining an antioxidant component that has been shown to effectively act on a wide range of reactive oxygen species.
[0011]
[Problems to be solved by the invention]
Under these circumstances, the present inventors have conducted a process of intensive research aiming at developing a composition for antioxidation of active oxygen species that effectively exhibits antioxidant activity against a wide range of reactive oxygen species. Focusing on the antioxidant HCDP contained in chicken extract and other animal extracts, anserine and carnosine, the amount of plant-derived antioxidant that matches the HCDP content while retaining the flavor and flavor of the extract itself Vitamin, which is a vitamin C and V. It has been found that by reinforcing E, it is possible to act on a wide range of active oxygens and to prevent biological proteins from being oxidatively decomposed by these active oxygens, thereby completing the present invention.
The present invention relates to an active oxygen species antioxidant composition having a specific antioxidant effect on reactive oxygen species such as a hydroxyl radical, a dichlorite radical and a peroxynitrate radical, and the antioxidant composition. It is an object of the present invention to provide an antioxidant food composition containing a compound.
[0012]
[Means for Solving the Problems]
The present invention for solving the above problems includes the following technical means.
(1) The anserine-carnosine mixture in an animal extract obtained by hot-water extraction from one or more muscles of edible poultry, livestock meat, and migratory fish may optionally have various content ratios of anserine and carnosine. The extract is mixed so as to be in a range of 3: 1 to 1: 3, and vitamin C (L-ascorbic acid or sodium L-ascorbate) is added to 20 to 100 mg of the anserine-carnosine mixture. 100 mg, vitamin E (α-tocopherol) in a ratio of 2.5 to 30 mg, having a specific antioxidant action on hydroxyl radical, dichlorite radical and peroxynitrate radical. The active oxygen species antioxidant composition.
(2) Anserine obtained as a permeate through a filtration membrane by subjecting an animal extract obtained by the above-mentioned antherin-carnosine mixture to hot water extraction to an ultrafiltration membrane having a molecular weight cutoff of 3,000 to 10,000. -The antioxidant composition according to the above (1), which is a carnosine mixture.
(3) The above-mentioned anserine-carnosine mixture is subjected to ultrafiltration membrane treatment of an animal extract obtained by the above-mentioned hot water extraction with a molecular weight cutoff of 3,000 to 10,000, and the filtrate permeated through the membrane is further fractionated. The antioxidant composition according to the above (1), wherein the composition is an anserine-carnosine mixture which is treated with a nanofiltration membrane or a reverse osmosis membrane having a molecular weight of 200 to 500 and is permeated on the filtration membrane and concentrated. .
(4) The above-mentioned anserine-carnosine mixture is subjected to ultrafiltration membrane treatment of the animal extract obtained by the above hot water extraction with a molecular weight cutoff of 3,000 to 10,000, and the filtrate permeated through the membrane is further fractionated. An anserine-carnosine mixture, which is treated with a nanofiltration membrane or a reverse osmosis membrane having a molecular weight of 200 to 500 and concentrated on the filtration membrane, and purified to a purity of 75% or more by a cation exchanger chromatograph. The antioxidant composition according to the above (1).
(5) An antioxidant food composition comprising the antioxidant composition according to any one of (1) to (4).
[0013]
BEST MODE FOR CARRYING OUT THE INVENTION
Next, the present invention will be described in more detail.
The present inventors, using an active oxygen actually generated in vivo, to evaluate whether the antioxidant obtained as a conventional food material effectively exhibits antioxidant activity in vivo, We examined how antioxidants act on the reactions that oxidatively degrade these animal proteins, and what their strength is.
A typical active oxygen generated in a living body is hydrogen peroxide (H 2 O 2 ), Hydroxyl radical (OH.), Dichlorite radical (ClO.), And nitric peroxide radical (ONOO.). Therefore, three kinds of OH., ClO. And ONOO. Having high ability to oxidatively decompose proteins in a test tube were prepared and subjected to a test. In comparison of antioxidant activity of each antioxidant, hemoglobin, ovalbumin, and serum albumin were used as target proteins. As a result, V.I. C, V. The antioxidant activity of E and anserine-carnosine varies greatly depending on the type of active oxygen. C gave to ONOO. It was found that E exhibited the strongest antioxidant activity against OH., And anserine-carnosine showed the strongest antioxidant activity against ClO. To further illustrate the characteristics of anserine and carnosine, it was found that anserine had stronger antioxidant properties against OH · than carnosine, and conversely, carnosine showed stronger antioxidant properties against ClO · than anserine. When the antioxidant activity was evaluated based on these results, as shown in Table 2 below, the antioxidant substance derived from animals, anserine-carnosine, was completely different from the evaluation by the DPPH method or the fluorescence emission method. Against the oxidative degradation of the animal proteins hemoglobin, ovalbumin, and bovine serum albumin. C and V. It was found to have almost the same antioxidant activity as E.
[0014]
The present inventors examined that the chicken extract contained anserine-carnosine in a ratio of 2: 1 to 4: 1, a beef extract of 1: 3 to 1: 5, and a pork extract of 1: 4. 1 : 1: 10 and tuna extract at a ratio of 10: 0 to 20: 1. These anserins and carnosine show the strongest antioxidant activity against ClO. And ONOO. Radicals, but OH. In the radical, V. Weaker than E; C has similar antioxidant activity to C. Anserin has a stronger antioxidant effect on OH · than carnosine, but carnosine has a stronger antioxidant effect on ClO · and ONOO · than anserine. From these facts, and from these facts, the content ratio of anserine and carnosine in the anserine-carnosine mixture in the HCDP can be arbitrarily adjusted to a range of 3: 1 to 1: 3 by mixing various extracts. , And V.V. for 100 mg of anserine-carnosine measured as a total amount. C, 20-100 mg; It was found that by adding E in a ratio of 2.5 to 30 mg, it was possible to almost completely prevent protein oxidation of these three strains by different active oxygens.
[0015]
In addition, even in the anserine-carnosine mixture at each purification step utilizing the taste and taste of the natural extract, if the HCDP content is adjusted, the same antioxidant activity as high-purity anserine-carnosine can be obtained, so that it was extracted from various materials. Even in the state of a natural extract, C and V. It is possible to produce an extract having high antioxidant activity by adding E. The anserine-carnosine content ratio in the natural extract according to the present invention that deviates from an appropriate range is, for example, pork extract and tuna, fish meat extract such as bonito, and pork has a high carnosine content and fish meat. The extract has a high anserine content. These extracts have an anserine and carnosine content ratio of 3: 1 to 1: 3, which is suitable for expressing antioxidant activity by mixing HCDP obtained from pork extract and fish meat extract approximately 1: 1. It is possible to do.
[0016]
Much has been reported on the usefulness of anserine-carnosine present in animal extracts as an antioxidant and on the potential of preventing lifestyle-related diseases. However, general practical use has not been advanced as much as vegetable antioxidants. This is because V.V. C and V. It showed much lower activity than antioxidant vitamins such as E. However, a large number of active oxygens are produced in the living body, which directly cause aging and disease, whereas the mode of action of antioxidants on these is unknown by conventional methods. According to the present invention, there can be obtained an antioxidant composition which strongly acts on three types of typical active oxygen, ClO., ONOO. And OH., Which are generated in a living body. Such an effect cannot be obtained. These are different from the results obtained by the conventional evaluation method and cannot be known by the conventional evaluation method. The composition of the present invention exhibits a specific and synergistic antioxidant effect on the active oxygen species when the above components are combined in a specific quantitative ratio. Has been demonstrated for the first time by carrying out the special experiments shown in the above-mentioned Test Examples and Examples, and the effects thereof can not be expected from conventional knowledge. For the purpose of preventing the occurrence of conventional lifestyle diseases, aging, and cancer, V.I. C and V. E, or when anserine and carnosine were used alone, none of them was effective against active oxygen generated in a living body, and was completely ineffective depending on the type of active oxygen. However, according to the composition of the present invention, an antioxidant activity can be effectively exerted on a wide variety of active oxygens, and thus, an antioxidant composition effective for prevention of novel lifestyle-related diseases and diseases which has never existed before. It can be prepared and provided.
[0017]
Next, the present invention will be described in detail with reference to test examples.
Test example 1
(1) Antioxidant activity of anserine-carnosine by a conventional method
The antioxidant peptides anserine and carnosine in the animal extract are combined with the plant antioxidant vitamin V. C and V. In order to compare the antioxidant activity of E, it was tested by the above-mentioned DPPH method and the chemiluminescence method (fluorescence emission method). Anserine / nitrate and carnosine used reagents (manufactured by Sigma), and antherin-carnosine mixture in animal extracts used chicken extract in each purification step. V. As C, L-ascorbic acid and sodium L-ascorbate reagent (manufactured by Wako Pure Chemical Industries, Ltd.) are used. As E, a superemulsion (trade name: Sun-Active VE-202, VE-702, manufactured by Taiyo Kagaku) having α-tocopherol emulsified in sucrose ester to impart hydrophilicity was used. The results were as shown in Table 1. In both the DPPH method and the fluorescence emission method, V.V. C shows the strongest antioxidant activity, E, the reagents anserin and carnosine, and the purified anserine-carnosine mixture were all measured as weak antioxidant activities.
[0018]
[Table 1]
Figure 2004231902
[0019]
(2) Antioxidant activity of anserine-carnosine in protein oxidative degradation system
Among the four typical types of active oxygen generated in the human body, H 2 O 2 Was difficult to judge because of its low oxidative degradation of protein, and was tested using three other OH., ClO. And ONOO. OH. Is the Fenton method (B. Halliwell et al., Analytical Biochemistry, 165, 215-219, 1987), ClO. Is sodium dichlorite, ONOO. Is the quenching flow reactor method (R. Radii et al. Journal of Biological Chemistry, 266, 4244-4250, 1991). The protein is dissolved in buffered physiological saline (pH 7.2) to a concentration of 2.5 mg / mL, and active oxygen (ClO: 400 ppm, OH :: 800 ppm) is added to the protein solution at a ratio of 10: 1 with respect to the protein solution. , ONOO .: 5 mM) and reacted at 37 ° C. for 30 minutes. The antioxidant was added at a ratio of 10: 1 to the protein solution 30 minutes before the addition of active oxygen. After electrophoresis of each reaction solution by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the stained gel was subjected to densitometer, or the protein reaction solution was directly subjected to GPC-HPLC of TSKG-3,000SW column. The proteolysis inhibitory activity was calculated by the following equation.
[0020]
Proteolysis inhibitory activity (%) = peak area of active oxygen-degrading protein when 5 mM of antioxidant is added x 100 (%) / peak area of undegraded protein
[0021]
Table 2 shows the results. From the results shown in Table 2, anserine and carnosine contained in various extracts have the strongest antioxidant activity against ClO. And ONOO. C shows antioxidant activity similar to anserine and carnosine; E was found to show the strongest antioxidant activity against OH.
[0022]
[Table 2]
Figure 2004231902
[0023]
(3) Inhibition of proteolytic degradation of active oxygen by compounding antioxidants
Anserine and carnosine contained in animal extracts such as chicken extract are V.V. C has a similar antioxidant activity to C. In contrast to E. Therefore, V. E is the strongest, and a mixture of animal-derived anserine-carnosine and V.E. C is the weakest against OH., And the animal-derived anserine-carnosine mixture is the strongest. These can be expected to have complementary effects on ClO.E, which has the weakest E. Further, in ONOO., V.E. C and V. Further complete antioxidant activity can be expected by adding E. The concentration of the anserine-carnosine mixture derived from various extracts was set to 1.2 mg / mL (about 5 mM). C sodium at 0.6 mg / mL (about 3.0 mM); Table 3 shows the proteolysis-inhibiting action when E was added and blended at 0.5 mg / mL (about 1 mM).
[0024]
[Table 3]
Figure 2004231902
[0025]
As shown in Table 3, the pork extract and the tuna extract had a V. C and V. The addition of E could not completely prevent the oxidative degradation by ClO. However, as shown in Table 4, it was considered that the content of anserine and carnosine was biased. From the results in Tables 3 and 4, the content ratio of anserine and carnosine for HCDP to exert appropriate antioxidant activity is in the range of the ratio of chicken extract and the ratio of beef extract. That is, anserine-carnosine was in the range of 3: 1 to 1: 3. When an equal amount of the UF-treated beef extract, the same pork extract, and the same tuna extract prepared in Example 3 were mixed, the content ratio of anserine and carnosine became almost equal to 1.8: 1.7. V. C and V. The rate of inhibition of oxidative decomposition with respect to ClO. In which E was added was 100%.
[0026]
[Table 4]
Figure 2004231902
[0027]
When the concentration of the anserine-carnosine mixture was set to 2.4 mg / mL (about 10 mM), V.V. C and V. When the added amount of E was 0.5 mg / mL (about 2.5 mM) and 0.06 mg / mL (about 0.15 mM), respectively, the results showed that the inhibitory activity against OH / oxidative degradation was 95% or more. . That is, although the antioxidant activity is increased by increasing the amount of the anserine-carnosine mixture derived from animal extract, the weight ratio of anserine-carnosine with respect to OH radicals that cannot completely suppress anserine-carnosine is reduced. V. C: 5: 1; When E was added at a ratio of 40: 1, the result that the protein degradation by various active oxygens was sufficiently suppressed was obtained. This means that V.V. per 100 mg of anserine-carnosine mixture. C is 20 mg; E becomes 2.5 mg. The upper limit is the recommended maximum daily intake of each antioxidant, so that anserine-carnosine and V.I. C is 3,000 mg; Since E is 300 mg, V.E. C is 100 mg; E amounts to 30 mg.
[0028]
(4) Glycoprotein production inhibitory action in diabetic rats (GK rats)
It is known that in the condition of diabetes, the amount of active oxygen in blood increases, and the amount of glycated protein, specifically glycated moglobin A1c, increases due to oxidized proteins and high glucose levels in blood. The production of glycated protein accompanying the increase in active oxygen means the accumulation of advanced glycation products (abbreviated as AGEs) in the living body, and various diseases associated with diabetes, such as diabetic hypertension, arteriosclerosis, The nephropathy, dementia, peripheral circulatory disorders, and the like are considered to be caused by the accumulation of AGEs. GK rats (Y. Ihara et al., FEBS Letters, 473, 24-26, 2000), a model animal of type 2 diabetes caused by overnutrition, are related to the present invention. C and V. It was tested whether the anserine-carnosine mixture to which E was added actually had an inhibitory effect on the production of glycated hemoglobin A1c promoted by the oxidation reaction in the living body. As shown in FIG. 3, 200 mg of UF-treated chicken extract anserine-carnosine per 1 kg body weight of GK rat, C 100 mg, V.I. When E20 mg was orally administered, the above Y. V. Ihara et al. When the feed containing 200 mg of anserine-carnosine was administered, the glycated hemoglobin A1c value was clearly reduced as compared with the case where 20 mg of E alone was administered.
[0029]
【Example】
Next, the present invention will be specifically described based on examples, but the present invention is not limited to the following examples.
Example 1
40 L of chicken extract obtained by hot water extraction from whole chicken was cut off with a molecular weight cut off of 3,000.
And applied to an ultrafiltration membrane (SEP-1013, manufactured by Asahi Kasei Corporation) to collect 32 L of the filtrate. The filtrate was concentrated under reduced pressure to a Brix concentration of 20%. The concentrated chicken extract had an anserine-carnosine content of 180 mg / g per solid. C (food additive) at 90 mg / g; E (Sunactive VE-202, manufactured by Taiyo Kagaku) was added to 18 mg / g, and dextrin (food additive) was added to 33% (1: 2) per solid of the concentrated chicken extract. By spray drying, about 30 g of antioxidant chicken extract powder was prepared. This powder had 120 mg of anserine-carnosine per 1 g of the powder. C, 60 mg; E contained 12 mg, and when orally administered to the above-mentioned GK rat for 2 weeks, as shown in FIG. 3, it had an activity of lowering blood glycated hemoglobin content.
[0030]
Example 2
1,200 L of the chicken extract extracted with hot water from a round chicken was applied to an ultrafiltration membrane having a molecular weight cut-off of 3,000 (SEP-3013 manufactured by Asahi Kasei), and 200 L of the filtrate permeated through the membrane was subjected to a molecular weight cut-off of 500 (NFT manufactured by Danish Separation Systems). Concentration was performed using the nanofiltration membrane of (-50) to prepare 20 L of chicken extract having a Brix concentration of 10%. Since this nano-filtered chicken extract contains 200 mg of anserine-carnosine per 1 g of solid, V.V. C and V. E was added and powdered by a freeze-drying method. Also, 20 L of the nano-filtered concentrated chicken extract was once desalted by adding water for production, 4 L of 0.5 M sodium phosphate buffer was added, and Dowex 50WX-8 equilibrated with 0.1 M phosphate buffer, pH 5.0. The solution was passed through a cation exchange resin column, anserine-carnosine was adsorbed on the column, the column was washed with a dilute buffer, and then anserine-carnosine was eluted and recovered with an alkaline solution. The solution was concentrated under reduced pressure, decolorized with activated carbon, and anserine-carnosine was precipitated by an ethanol precipitation method, and the alcohol was removed under reduced pressure to prepare about 10 g of a purified anserine-carnosine mixture powder. The anserine-carnosine content ratio of this purified powder was 1.2: 1.0, the total amount of anserine-carnosine was 500 mg per 100 mL of 2% lemon juice, and V.V. C 250 mg, V.I. E was added to be 30 mg to prepare an antioxidant soft drink.
[0031]
Example 3
3 L of water was added per 1 kg of deboned pork, beef, and tuna meat, extracted with hot water at 98 ° C. for 1 hour, and the oil layer was separated with a separating funnel, followed by suction filtration using diatomaceous earth as a filter aid. These extracts contained anserine and carnosine as a mixture, and the content of anserine-carnosine per 1 g of solid was 96 mg for pigs, 67 mg for cows, and 120 mg for tuna. These extracts were concentrated under reduced pressure to Brix 5.0%, and three kinds were mixed in equal amounts. Since the anserine-carnosine content ratio at this time was 1.8: 1.7, V.V. C sodium (food additive) 50 mg; E (manufactured by Taiyo Kagaku, Sunactive VE720) was added in an amount of 20 mg and sterile-filtered to prepare a liquid animal extract. In addition, these diatomaceous earth-filtered extracts were passed through an ultrafiltration membrane (SEP-1013 manufactured by Asahi Kasei) to remove proteins, concentrated under reduced pressure, and mixed in the same manner as described above. C sodium 50 mg and V.I. E was added in an amount of 30 mg, and a liquid antioxidant animal extract was similarly prepared. These extracts exhibited activities to almost completely suppress the degradation of proteins by the three types of active oxygen shown in Table 3.
[0032]
【The invention's effect】
As described in detail above, the present invention relates to an antioxidant composition comprising a specific component having an antioxidant ability derived from an animal natural extract in a specific quantitative ratio, and according to the present invention, The following effects are obtained.
(1) It is possible to provide the active oxygen species antioxidant composition having an antioxidant action specific to various reactive oxygen species.
(2) It is possible to provide a novel antioxidant composition having antioxidant performance against all three types of representative active oxygen generated in a living body.
(3) Conventionally, C and V. It is possible to provide an antioxidant composition for a wide range of reactive oxygen species containing anserine and carnosine as active ingredients, which has been considered to exhibit only extremely lower activity than antioxidant vitamins such as E.
(4) anserine-carnosine mixture; C, and V.I. By combining E, a synergistic antioxidant action that cannot be expected with these components alone can be obtained.
(5) From these, it is possible to prepare an antioxidant composition or food that can effectively prevent the development of lifestyle-related diseases and other diseases involving active oxygen.
[Brief description of the drawings]
FIG. 1 shows the inhibitory effects of various antioxidants against oxidative degradation of ovalbumin by typical active oxygen generated in a living body (A: ClO., B: OH, C: ONOO., 1: undegraded ovalbumin, 2: degraded ovalbumin, 3: added VC, 4: added VE, 5: added anserine, 6: added carnosine, 7: added purified anserin-carnosine derived from chicken extract).
FIG. 2 shows the inhibitory effect of the substance of the present invention on the production of glycated hemoglobin A1c in blood in GK rats of a diabetes disease state model (CE + VC + VE: UF-treated chicken extract + vitamin C + vitamin E; CE + VC: UF-treated chicken extract + vitamin) C; VE: vitamin E alone).

Claims (5)

食用家禽類、畜肉類、及び回遊性魚類の1種以上の筋肉より熱水抽出して得られる動物性エキス中のアンセリン−カルノシン混合体の、アンセリンとカルノシンの含量比率を任意に各種エキスを混合することによって、3:1〜1:3の範囲になるように調整し、該アンセリン−カルノシン混合体100mgに対してビタミンC(L−アスコルビン酸又はL−アスコルビン酸ナトリウム)を20〜100mg、ビタミンE(α−トコフェロール)を2.5〜30mgの割合で配合したことを特徴とする水酸化ラジカル、ジ亜塩素酸ラジカル及び過酸化硝酸ラジカルに対して特異的な抗酸化作用を有する当該活性酸素種抗酸化用組成物。Various extracts of anserine-carnosine mixture in an animal extract obtained by hot water extraction from one or more muscles of edible poultry, livestock meat, and migratory fish, with anserine and carnosine mixed at will. To adjust the range of 3: 1 to 1: 3, 20 to 100 mg of vitamin C (L-ascorbic acid or sodium L-ascorbate) to 100 mg of the anserine-carnosine mixture, E (α-tocopherol) compounded in a ratio of 2.5 to 30 mg, the active oxygen having a specific antioxidant action against a hydroxyl radical, a dichlorite radical and a peroxynitrate radical. Species antioxidant composition. 上記アンセリン−カルノシン混合体が、上記熱水抽出して得られる動物エキスを分画分子量3,000〜10,000の限外ろ過膜処理して、ろ過膜透過画分として得られるアンセリン−カルノシン混合体であることを特徴とする請求項1記載の抗酸化用組成物。The anserine-carnosine mixture obtained by subjecting the animal extract obtained by the hot water extraction to an ultrafiltration membrane having a molecular weight cut-off of 3,000 to 10,000 is treated with the anserine-carnosine mixture to obtain a permeation fraction of the filtration membrane. The antioxidant composition according to claim 1, which is a body. 上記アンセリン−カルノシン混合体が、上記熱水抽出して得られる動物エキスを分画分子量3,000〜10,000の限外ろ過膜処理して、該ろ過膜透過液を更に分画分子量200〜500のナノろ過膜もしくは逆浸透膜で処理してろ過膜上に透過阻止されて濃縮されたアンセリン−カルノシン混合体であることを特徴とする請求項1記載の抗酸化用組成物。The anserine-carnosine mixture is treated with an ultrafiltration membrane having a molecular weight cut-off of 3,000 to 10,000 for the animal extract obtained by the hot water extraction, and the filtrate permeated through the filter is further subjected to a molecular weight cut-off of 200 to 10,000. 2. The antioxidant composition according to claim 1, wherein the composition is an anserine-carnosine mixture which is treated with a nanofiltration membrane or a reverse osmosis membrane of 500 to prevent permeation on the filtration membrane and is concentrated. 上記アンセリン−カルノシン混合体が、上記熱水抽出して得られる動物エキスを分画分子量3,000〜10,000の限外ろ過膜処理して、該ろ過膜透過液を更に分画分子量200〜500のナノろ過膜もしくは逆浸透膜で処理してろ過膜上に濃縮されたものを陽イオン交換体クロマトグラフによって純度75%以上に精製されたアンセリン−カルノシン混合体であることを特徴とする請求項1記載の抗酸化用組成物。The anserine-carnosine mixture is treated with an ultrafiltration membrane having a molecular weight cut-off of 3,000 to 10,000 for the animal extract obtained by the hot water extraction, and the filtrate permeated through the filter is further subjected to a molecular weight cut-off of 200 to 10,000. An anserine-carnosine mixture which has been treated with a 500 nanofiltration membrane or a reverse osmosis membrane and concentrated on the filtration membrane to be purified to a purity of 75% or more by cation exchanger chromatography. Item 7. An antioxidant composition according to Item 1. 請求項1から4のいずれかに記載の抗酸化用組成物を配合したことを特徴とする抗酸化性食品組成物。An antioxidant food composition comprising the antioxidant composition according to any one of claims 1 to 4.
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WO2006115153A1 (en) * 2005-04-25 2006-11-02 Ajinomoto Co., Inc. Food composition having skin condition-improving effect
JP2007181421A (en) * 2006-01-06 2007-07-19 Yaizu Suisankagaku Industry Co Ltd Fish and shellfish extract highly containing imidazole dipeptide, food and drink containing imidazole dipeptide, and method for producing the fish and shellfish extract highly containing imidazole dipeptide
JP2009046451A (en) * 2007-08-22 2009-03-05 Tokai Bussan Kk Method for producing antioxidant dipeptide
US7727961B2 (en) 2004-04-30 2010-06-01 Tokai Bussan Co., Ltd. Antioxidant constituents
JP2010235503A (en) * 2009-03-31 2010-10-21 Yaizu Suisankagaku Industry Co Ltd Method for producing imidazole dipeptide-containing composition
JP2016158549A (en) * 2015-02-27 2016-09-05 ポッカサッポロフード&ビバレッジ株式会社 Lemon juice-containing beverage, production method thereof, and acidity modification method
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US7727961B2 (en) 2004-04-30 2010-06-01 Tokai Bussan Co., Ltd. Antioxidant constituents
JP2006265161A (en) * 2005-03-23 2006-10-05 Japan Research & Development Association For New Functional Foods Antioxidant composition
WO2006115153A1 (en) * 2005-04-25 2006-11-02 Ajinomoto Co., Inc. Food composition having skin condition-improving effect
JP2007181421A (en) * 2006-01-06 2007-07-19 Yaizu Suisankagaku Industry Co Ltd Fish and shellfish extract highly containing imidazole dipeptide, food and drink containing imidazole dipeptide, and method for producing the fish and shellfish extract highly containing imidazole dipeptide
JP4612549B2 (en) * 2006-01-06 2011-01-12 焼津水産化学工業株式会社 Imidazole dipeptides-rich seafood extract, imidazole dipeptides-containing food and drink, and method for producing imidazole dipeptides-rich seafood extract
JP2009046451A (en) * 2007-08-22 2009-03-05 Tokai Bussan Kk Method for producing antioxidant dipeptide
JP2010235503A (en) * 2009-03-31 2010-10-21 Yaizu Suisankagaku Industry Co Ltd Method for producing imidazole dipeptide-containing composition
JP2016158549A (en) * 2015-02-27 2016-09-05 ポッカサッポロフード&ビバレッジ株式会社 Lemon juice-containing beverage, production method thereof, and acidity modification method
WO2020085459A1 (en) * 2018-10-26 2020-04-30 国立大学法人東京大学 Antioxidation agent, antiglycation agent or anti-inflammatory agent
JP7464945B2 (en) 2018-10-26 2024-04-10 国立大学法人 東京大学 Antioxidants, antiglycation agents or anti-inflammatory agents

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